This laboratory focuses on elucidating the coupling of forces, structure, and dynamics of biologically important macromolecules. The next challenge in structural biology is to understand the physics of interactions between molecules in aqueous solution. The ability to take advantage of the increasing number of protein and nucleic acid structures determined by x-ray crystallography and solution NMR will depend critically on this knowledge, both to understand the strength and specificity of interactions among biologically important macromolecules that control cellular function and to design rationally agents that can effectively compete with those specific interactions associated with disease. Our past results have shown that experimentally measured forces at close approach are very different from those predicted by current, conventionally accepted theories of intermolecular interactions. We have interpreted the observed forces as indicating the dominating contribution from water structuring energetics. The research program consists of two parts. We use osmotic stress and x-ray scattering to measure directly forces between biological macromolecules in macroscopic condensed arrays. Second, we measure changes in hydration accompanying specific recognition reactions of biologically important macromolecules in dilute solution. This is a first step toward linking our observation of dominating hydration forces between surfaces in condensed arrays to the interaction of individual molecules in solution. During this past year, we investigated the role of small solutes in the assembly of DNA by spermidine. Contrary to conventional expectations, we find that alcohol enhanced precipitation of DNA is not facilitated by a lowered dielectric constant and increased electrostatic attraction, but rather because alcohols are excluded from DNA. We were able to map the spatial distribution function of two alcohols from the DNA surface from the sensitivity of interhelical spacing measured by x-ray diffraction to alcohol concentration using the osmotic stress technique. The concentration profile is exponential with a 0.3-0.4 nm decay length characteristic of a repulsive hydration force. Alcohols are interacting with DNA through their effect on water structuring. For homologous alcohols the magnitude of the repulsion seems to scale with size. 2-Methyl-2,4-pentanediol is twice the size of i-propanol and is also twice as excluded. Since glycerol, however, is intermediate in size between these two alcohols, but is not observably excluded, size enters as an interaction summation over the chemical moieties of the solute rather than as a steric crowding. The polar solute betaine glycine inhibits spermidine-induced assembly. In this case, an exponential increase in betaine concentration as helices move closer is observed, once again with the 0.3-0.4 nm decay length characteristic of hydration forces. In contrast to alcohols, these are attractive water structuring forces rather than repulsive. We can take advantage of this inclusion to probe DNA-DNA attractive forces. In essence, betaine applies outward pressure on the DNA assembly that is resisted by spermidine-mediated attraction. Quite surprisingly, we find that there is a transition to a much weaker attractive state as betaine concentration increases. We have long suspected that DNA assembly is accompanied a loss in entropy as bound counterions are localized to maximize attraction. Indeed, interplay between attractive forces and motional constraints or ordering is a common feature of many recognition reactions. We are currently investigating if the transition is caused by an abrupt change from correlated or ordered binding of spermidine to an uncorrelated or disordered binding. We are now planning experiments to extract the energies and entropies involved with this process. We have continued investigating the role of water in the sequence specific recognition reaction of the restriction nuclease EcoRI. The exceptionally stringent specificity of this enzyme is a paradigm for recognition reactions. We measure the number of waters coupled to a binding reaction from the dependence of the binding free energy on water chemical potential or, equivalently, osmotic pressure. We have previously found that nonspecific complex of the protein sequesters some 110 more waters than the specific one and that this water is likely at the protein-DNA interface. We have further shown that the dissociation rate of the specific complex is also very sensitive to osmotic pressure requiring the net binding of 120-150 waters. During this past year we have investigated the temperature sensitivity of the ratio of specific and nonspecific binding constants directly measured using a competition assay. We found that the difference of 110 waters between the two complexes is temperature independent. We further found that the heat capacity change between specific and nonspecific binding is only about half of that found for specific binding alone. Since changes in heat capacity in binding reactions are generally ascribed to the release of structured water, the water trapped at the protein-DNA interface of the nonspecific complex is likely less ordered than the water bound to the two isolated surfaces. At low osmotic stresses 110 sequestered waters are found even for complexes with noncognate sequences that differ by only a single base pair from the specific recognition sequence. At least some of this water, however, can be removed from these noncognate complexes by applying high enough osmotic pressures. We are only able to remove water from complexes with sequences that differ by one base pair. The amount of water we are able to remove from a noncognate complex correlates with the ability of the enzyme to cleave the sequence. Water and function seem closely connected.